Fundamental to the response of cells to external stimuli is the stimulation of cell surface receptors by external signals. While there are a number of different receptors embedded within the plasma membrane, and a variety of such external signals, e.g., hormones, blood and growth factors, neurotransmitters, and radiation of a specific wavelength, there is a limited number of internal signals or second messengers employed within the cell. A second messenger is one that activates an appropriate cellular response to a specific external signal. It becomes activated when the receptor stimulated by an external signal excites an internal enzyme, which in turn stimulates the production of a second messenger substance.
An early signal transduction system identified in the art was the beta adrenergic receptor-adenylate cyclase pathway. This system employs the second messenger cyclic adenosine monophosphate (cAMP), a derivative of adenosine triphosphate (ATP). Its mechanism of action is now understood to proceed as follows: the external signal-receptor complex interacts with a guanosine nucleotide binding protein called a G-protein. G-protein activates adenylate cyclase, which in its activated form can catalyze the production of the second messenger, cAMP, from ATP. cAMP, in turn, causes cellular activity, e.g., protein synthesis, secretion, cytoskeletal movement, constituting a cellular response.
G-proteins are a class of regulatory proteins which bind guanosine di- and triphosphate nucleotides, i.e., GDP and GTP, respectively. The family of G-proteins serves as peripherally membrane-bound signal transducing polypeptides (STPs), coupling activation of cell surface receptors to the regulation of intracellular effectors. These proteins can activate the enzymatic abilities of adenylate cyclase or a phosphodiesterase while binding GTP. Examples of known and probable G-proteins include G.sub.s and G.sub.i, which are responsible for the regulation of adenylate cyclase; transducin, which activates a cGMP-specific phosphodiesterase in the retina; ADP-ribosylation factor (ARF) in the liver (Kahn et al. J. Biol. Chem. 259:6228-6234, 1984); and P21, the product of the ras protooncogene. (For a review, see Whitman et al., Phosphoinositides and Receptor Mechanisms, copyright 1986 by Alan R. Liss, Inc. pp. 197-217).
A second signal transduction system serves as a basis for cellular signalling by mitogenic growth factors such as growth hormone (GH), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and radiation of specific wavelengths. This system involves various intermediates in the inositol metabolic pathway. It employs calcium ions and a combination of second messengers ultimately derived from phosphatidylinositol (PI), which is a minor plasma membrane constituent. In this system an external signal, such as light in the photoreceptor cell, activates a receptor, e.g., rhodopsin, which then, by means heretofore unknown, stimulates the catalytic activity of phospholipase-C. A key event with regard to the second messenger function is the hydrolysis of an inositol derivative, phosphatidylinositol 4,5-biphosphate (PIP.sub.2), a minor membrane constituent, by phospholipase-C (PL-C) to yield inositol-1,4,5-trisphosphate (IP.sub.3) and diacylglycerol (DG). Both of these reaction products act as second messengers in at least two different systems: DG controls ion currents through the membrane by regulating membrane permeability to various ions and the activity of protein kinase C; while IP.sub.3 regulates the concentration of intracellular Ca.sup.+2 which in turn affects many cellular processes, e.g., cell division and proliferation.
Because of the intimate involvement of the inositol metabolic pathway in this second messenger system, it is understood that failure in the pathway mediate the development of a number of disease states. For example, there is now a large body of evidence supporting the concept that the secondary effects of diabetes, i.e., vascular degeneration and slowed nerve conduction, are the result of stepped-up sorbitol production that results from a failure of the inositol metabolic pathway. The effect of chronically high blood sugar levels on the inositol pathway is to retard inositol metabolism. This may be the result of the inactivity of a regulatory G-type protein due to the glycosylation of nuclear elements, e.g., genes or regulatory proteins, or be the result of the direct glycosylation of the G-type regulatory protein.
Retinitis pigmentosa, a disease of the eye characterized by toxic levels of unmetabolized GTP in photoreceptors, may result from a failure of GTP hydrolysis, due to absent or reduced levels of GTPase activity of a G-protein, or by the inability of a mutated G-protein to bind or mediate hydrolysis of GTP.
Current evidence suggests that at least some types of human cancer, or uncontrolled cell proliferation, are the result of a mutation in a regulatory enzyme of the inositol system. The suspected mutation is understood to prevent the hydrolysis of GTP, the inactivating step for the entire inositol metabolic pathway, including systems initiated by growth hormone (GH). If the inositol system is unable to shut off, the result is uncontrolled cell division, or malignancy. Alternatively, the malignant state could result from the hyperproduction of IP-3 caused by the overproduction, or faulty production, of an enzyme controlling the inositol pathway.
Disease states characterized by the lack of cell division, i.e. lack of proliferation, can also be the result of a failure in the inositol-related signal transduction system to increase intracellular Ca.sup.+2 levels, or to respond to GH or other growth factors.
Accordingly, the elucidation of the regulatory mechanism involved in the inositol-related signal transduction system will provide a better understanding of the disease states which result from its dysfunction, and can lead to the development of Preventative and/or compensatory measures. More specifically, there exists a need for methods of treating disease states resulting from the dysfunction of this system, and for methods of regulating inositol metabolism in cultured cells and cells of higher organisms.
Therefore, it is an object of this invention to provide proteins linking functionally cell membrane receptors and the inositol-related signal transducing system. It is also an object to provide a method of regulating the inositol metabolic pathway to compensate for disease states resulting from its dysfunction. Another object is to provide a method of stimulating and of depressing the inositol metabolic pathway in cell cultures and multicellular organisms.
These and other objects of the invention will be apparent from the description, drawing, and claims which follow.